This invention relates to new kinds of cation-exchangers that adsorb/bind substances at unusually high levels of ionic strengths. These cation-exchangers enable new ways for removing positively charged substances, for instance bioorganic substances, from liquids that preferably are aqueous.
Cation-exchangers comprise a plurality of ligands carrying a net negative charge. These kinds of ligands shall hereinafter be called “cation-exchange ligands”. They include a possible spacer between the support matrix and the part of the ligand interacting with the substance to be bound. Cation-exchange ligands as contemplated in the context of the present invention typically have a molecular weight <1000, such as <700 daltons excluding the molecular weight contribution of halo groups that may be present.
The ligands are bound to a suitable carrier material, which typically is insoluble or insolubilizable in aqueous liquid media. Insoluble carrier materials will hereinafter be referred to as matrices and include also insolubilized forms or insolubilzable carrier materials.
The term “bimodal”, in the context of this invention, refers to a ligand that is capable of providing at least two different, but co-operative, sites which interact with the substance to be bound. One of these sites gives an attractive type of charge-charge interaction between the ligand and the substance of interest. The second site gives hydrogen-bonding and/or hydrophobic interactions. Other kinds of interactions may also be present, for instance π—π, charger transfer and induced dipole interaction. There may also be present other sites giving rise to interactions with the ligand and the substance of interest.
The term remove/removal or separate/separation in the context of the present invention will encompass removal of a substance for any purpose, thus including adsorption to a cation-exchanger for isolation, purification, concentration, analysis etc. Removal/separation of impurities from a liquid will thus be included. In this case the liquid can be further processed with respect to some other substance(s) that is(are) of interest. The adsorbed substance may also be further processed. In this latter case the substance is typically de-sorbed and collected. If needed the substance is subjected to further purification steps. Good process economics requires that the cation-exchanger is regenerated and re-used after de-sorption.
Disadvantages With Earlier Techniques
Cation-exchange adsorption has for many years been of interest in large scale processing of fermentation broths and the like. These kinds of liquids typically have a high ionic strength making them unsuitable for direct application to conventional ion-exchangers. One reason has been that conventional ion exchangers adsorb proteins and other bio-polymers only at moderate ionic strengths, for instance at 0.1 M or lower in NaCl. This has implied dilution of process liquids giving large volumes to process and heavy investments in process equipment.
Related Publications
WO 9965607 (Amersham Pharmacia Biotech AB) discloses cation-exchangers in which there are linear cation-exchange ligands —A—X—Y(—Z)n where n is an integer ≧1, A is a spacer, X is —O—, —SR′— or —N(R′)(R″) (R′ and R″ are H, a free electron pair and certain groups providing a carbon directly attached to the heteroatom), Y is certain hydrocarbyl groups with the disclaimer that some of them shall not be combined with X being —O— or —S—, and finally Z is a cation-exchange group. The invention described in WO 9965607 is based on the discovery that in the defined group of ligands, there are cation-exchange ligands that, in contrast to conventional cation-exchangers, require elution ionic strengths that are up to 200% compared to a reference sulphopropyl cation-exchanger. It is speculated that there may be found extreme ligands that require ionic strengths more than 200% of the reference cation-exchanger.
WO 9808603 (Upfront Chromatography) discloses separation media of the general structure M-SP1-L in which M is a support matrix that may be hydrophilic, SP1 is a spacer and L comprises a mono- or bicyclic homoaromatic or heteroaromatic moiety that may be substituted (a homoaromatic moiety comprises an aromatic ring formed only by carbon atoms). In one variant L is X—A-SUB where X is —O—, —S— or —NH— and A is the homoaromatic or heteroaromatic moiety that is substituted. The substituent on A may be an acidic group which means that —SP1-X—A-SUB can be a cation-exchange ligand which is linear. The separation medium is suggested for the adsorption of proteins, in particular immunoglobulins, by hydrophobic interactions rather than cation-exchange (salt concentration up to 2 M).
WO 9600735 and WO 9609116 (Burton et al) disclose ion exchange resins in which the hydrophobicity/hydrophilicity of the resin including the ligand is changed upon changing in pH. The hydrophobicity may also be increased synthetically by the introduction of hydrophobic non-ionizable ligands. Adsorption/desorption is controlled by altering the hydrophobicity/hydrophilicity of the matrix including the ligand, for instance by changing the pH.
U.S. Pat. No. 5,789,578 (Burton et al) suggests to immobilise a thiol containing ligand, such as 3-mercaptopropionic acid, by addition of the thiol group over carbon-carbon double bond attached to a support matrix. The inventors in this case neither employ nor suggest the use of the material obtained for cation-exchange adsorptions.
WO 9729825 (Amersham Pharmacia Biotech AB) discloses anion exchangers in which the anion exchanging ligands comprises oxygen and/or nitrogens at a distance of 2–3 carbon atoms from the nitrogen atom of a primary, secondary or tertiary ammonium group (positively charged, cationic).
Dipolar adsorbents prepared by coupling of sulphanilic acid using epichlorohydrin has been described (ligand+spacer=—CH2CHOHCH2N+H2C6H4SO3−) (Porat et al., J. Chromatog. 51 (1970) 479–489; and Ohkubo et al., J. Chromatog. A, 779 (1997), 113–122). The articles do not disclose a method in which the ligand is negatively charged and the substance to be removed is positively charged.
2,4,6-trihalo-1,3,5-triazine has been utilized to bind different compounds RHNR′X to carriers inter alia to cellulose. R has been hydrogen, aryl or alkyl, R′ alkylene or arylene and X carboxy, sulphonyl, phosphate, phosphonate, boronate, etc. (See Behrend et al., WPI Abstract Accession No. 86-312313 (=DD-A-237844). This coupling methodology gives structures that are unstable to hydrolysis.
EP 326233 discloses a cation-exchanger in which there is a hydrophobic support matrix to which cation exchanging groups are attached. The hydrophobicity makes this type of cation-exchangers unsuitable for separation of biomolecules such as proteins.